Occurrence of a 16SrII group phytoplasma associated with crotalaria witches' broom in Hainan, China

Crotalaria (Fabaceae), commonly know as rattlepods or rattlebox, is a genus of herbaceous plants and woody shrubs indigenous to the tropics and subtropics. More than 600 species of Crotalaria worldwide are described and about 36 are found in China. Most species of Crotalaria are usually used as cover crops or green manure so as to improve soil properties and recycle plant nutrients. Crotalaria contains pyrrolizidine alkaloid, a serious liver toxin, and it has been shown to be poisonous to most livestock.

In 2006, symptoms suggestive of phytoplasma infection were observed on over 20 Crotalaria species in the germplasm garden of genus Crotalaria, which is located at the Chinese Academy of Tropical Agricultural Science, Hainan, China. The plants showed much proliferation of little leaves and shoots with shortened internodes (Fig.1, Fig.2).

Healthy and infected leaf samples from two Crotalaria species, Crotalaria szemaoensis and Crotalaria zanzibarica infected most seriously, were collected from the garden and tested by PCR amplification for phytoplasma-specific 16SrDNA. Nucleic acids were extracted from fresh leaves as described by Parmessur et al. (2002) and used as the template in nested PCR reactions with primers P1/P7 and R16F2n/R2 (Lee et al., 1998). DNA from phytoplasma infected periwinkle was used as a positive control. DNA fragments of the expected sizes (about 1.8 kb and 1.2 kb respectively) were obtained from the infected plant samples but not from the apparently healthy one. The PCR products (1.8kb) were cloned and sequenced (GenBank Accession No. EF656453, EF656454). Using a multiple alignment program (DNASTAR Version 6.13), the sequences showed that the phytoplasmas from the two Crotalaria species were 99.9% identical to each other. They had most similarity to members of the 16SrII group of phytoplasmas, with 100% identity to the phytoplasmas from Sweet potato witches' broom (DQ452417) and Pear decline phytoplasma (EF193157), and 98.5% to 98.7% similarity to the phytoplasma from Sunhemp (Crotalaria juncea) witches' broom (X76433) (Seemüller et al., 1994) (Fig. 3). To our knowledge, this is the first identification of a phytoplasma from the peanut witches' broom group infecting C. szemaoensis and C. zanzibarica in China.